Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections

Abstract

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.

Figure 1.

Relationship between parasite density and histidine-rich protein II (HRP2) concentration in IBSM studies. (A) HRP2 concentration and parasite density profiles over days after infection with parasitized red blood cells. Dashed line and empty squares represent parasite density; solid line and empty circles represent HRP2 concentration. The means of results from 16 subjects are shown. (B) Relationship between parasite density and HRP2 concentration in the 0–5 parasites per µL range pre-treatment infection.

Figure 2.

Distribution of specimens by parasite density and histidine-rich protein II (HRP2) concentration in induced blood-stage malaria challenge studies (A, D); Myanmar study (B, E); and Uganda study (C, F). (A–C). The outer clear bars represent the specimens that were positive by quantitative reverse transcription polymerase chain reaction; overlaid are the number of positive test results by ultra sensitive rapid diagnostic test (uRDT) (checkered bars) and by RDT (gray bars). (D–F). The outer clear bars represent the specimens that were positive by quantitative HRP2 assay; overlaid are the number of positive test results by uRDT (checkered bars) and by RDT (gray bars).

Figure 3.

Relationship between parasite density and histidine-rich protein II (HRP2) concentration shown as box plots for ranges of parasite densities. The data shown are for the Uganda specimens. The upper and lower bounds of the boxes represent the third and first quartiles, respectively. The mean is indicated by a solid line and the whiskers indicate the 95th and 5th percentiles. The leftmost bin for 0 parasites per µL (p/µL) corresponds to HRP2-positive but quantitative reverse transcription polymerase chain reaction-negative specimens. The descriptive statistics are provided in Table 3.

Figure 4.

Histogram of Uganda asymptomatic clinical specimens arranged by histidine-rich protein II (HRP2) concentration ranges and broken out into confirmed parasitemic (gray shaded bars) and non-parasitemic (spotted bars) specimens based on quantitative reverse transcription polymerase chain reaction data.

Figure 5.

Cumulative proportion of the confirmed parasitemic asymptomatic specimens in the Uganda study that are uRDT (solid line) or RDT (dashed line) positive with (A) decreasing parasite density as determined by quantitative reverse transcription polymerase chain reaction (left to right) and (B) decreasing histidine-rich protein II (HRP2) concentration as determined by the HRP2 Q-plex enzyme-linked immunosorbent assay (left to right).